Functional, immunological characterization, and anticancer activity of BaMtx: A new Lys49- PLA2 homologue isolated from the venom of Peruvian Bothrops atrox snake (Serpentes: Viperidae).

Bothorps atrox is responsible for most of the ophidism cases in Perú. As part of the envenoming, myotoxicity is one of the most recurrent and destructive effects. In this study, a myotoxin, named BaMtx, was purified from B. atrox venom to elucidate its biological, immunological, and molecular characteristics. BaMtx was purified using CM-Sephadex-C-25 ion-exchange resin and SDS-PAGE analysis showed a unique protein band of 13 kDa or 24 kDa under reducing or non-reducing conditions, respectively. cDNA sequence codified a 122-aa mature protein with high homology with other Lys49-PLA2s; modeled structure showed a N-terminal helix, a ß-wing region, and a C-terminal random coil. This protein has a poor phospholipase A2 enzymatic activity. BaMtx has myotoxic (DMM = 12.30 ± 0.95 µg) and edema-forming (DEM = 26.00 ± 1.15 µg) activities. Rabbit immunization with purified enzyme produced anti-BaMtx antibodies that reduced 50.28 ± 10.15% of myotoxic activity and showed significant cross-reactivity against B. brazili and B pictus venoms. On the other hand, BaMtx exhibits mild anti-proliferative and anti-migratory effects on breast cancer cells, affecting the ROS and NADH levels, which may reduce mitochondrial respiration. These results contribute to the understanding of B. atrox Lys49-PLA2 effects and establish the anticancer potential de BaMtx.

Fibrinogen-clotting enzyme, pictobin, from Bothrops pictus snake venom. Structural and functional characterization.

A thrombin-like enzyme, pictobin, was purified from Bothrops pictus snake venom. It is a 41-kDa monomeric glycoprotein as showed by mass spectrometry and contains approx. 45% carbohydrate by mass which could be removed with N-glycosidase. Pictobin coagulates plasma and fibrinogen, releasing fibrinopeptide A and induces the formation of a friable/porous fibrin network as visualized by SEM. The enzyme promoted platelet aggregation in human PRP and defibrination in mouse model and showed catalytic activity on chromogenic substrates S-2266, S-2366, S-2160 and S-2238. Pictobin interacts with the plasma inhibitor α2-macroglobulin, which blocks its interaction with fibrinogen but not with the small substrate BApNA. Heparin does not affect its enzymatic activity. Pictobin cross reacted with polyvalent bothropic antivenom, and its deglycosylated form reduced its catalytic action and antivenom reaction. In breast and lung cancer cells, pictobin inhibits the fibronectin-stimulated migration. Moreover, it produces strong NADH oxidation, mitochondrial depolarization, ATP decrease and fragmentation of mitochondrial network. These results suggest by first time that a snake venom serinprotease produces mitochondrial dysfunction by affecting mitochondrial dynamics and bioenergetics. Structural model of pictobin reveals a conserved chymotrypsin fold ß/ß hydrolase. These data indicate that pictobin has therapeutic potential in the treatment of cardiovascular disorders and metastatic disease.

Biochemical and molecular characterization of the hyaluronidase from Bothrops atrox Peruvian snake venom.

Snake venoms are a rich source of enzymes such as metalloproteinases, serine proteinases phospholipases A2 and myotoxins, that have been well characterized structurally and functionally. However, hyaluronidases (E.C.3.2.1.35) have not been studied extensively. In this study, we describe the biochemical and molecular features of a hyaluronidase (Hyal-Ba) isolated from the venom of the Peruvian snake Bothrops atrox. Hyal-Ba was purified by a combination of ion-exchange and gel filtration chromatography. Purified Hyal-Ba is a 69-kDa (SDS-PAGE) monomeric glycoprotein with an N-terminal amino acid sequence sharing high identity with homologous snake venom hyaluronidases. Detected associated carbohydrates were hexoses (16.38%), hexosamines (2.7%) and sialic acid (0.69%). Hyal-Ba selectively hydrolyzed only hyaluronic acid (HA; specific activity = 437.5 U/mg) but it did not hydrolyze chondroitin sulfate or heparin. The optimal pH and temperature for maximum activity were 6.0 and 40 °C, respectively, and its Km was 0.31 µM. Its activity was inhibited by EDTA, iodoacetate, 2-mercaptoethanol, TLCK and dexamethasone. Na+ and K+ (0.2 M) positively affect hyaluronidase activity; while Mg2+, Br2+, Ba2+, Cu2+, Zn2+, and Cd2+ reduced catalytic activity. Hyal-Ba potentiates the hemorrhagic and hemolytic activity of whole venom, but decreased subplantar edema caused by an l-amino acid oxidase (LAAO). The Hyal-Ba cDNA sequence (2020 bp) encodes 449 amino acid residues, including the catalytic site residues (Glu135, Asp133, Tyr206, Tyr253 and Trp328) and three functional motifs for N-linked glycosylation, which are conserved with other snake hyaluronidases. Spatial modeling of Hyal-Ba displayed a TIM-Barrel (α/ß) fold and an EGF-like domain in the C-terminal portion. The phylogenetic analysis of Hyal-Ba with other homologous Hyals showed the monophyly of viperids. Further, Hyal-Ba studies may extend our knowledge of B. atrox toxinology and provides insight to improve the neutralizing strategies of therapeutic antivenoms.

Biochemical, biological and molecular characterization of an L-Amino acid oxidase (LAAO) purified from Bothrops pictus Peruvian snake venom.

An L-amino acid oxidase from Peruvian Bothrops pictus (Bpic-LAAO) snake venom was purified using a combination of size-exclusion and ion-exchange chromatography. Bpic-LAAO is a homodimeric glycosylated flavoprotein with molecular mass of ∼65 kDa under reducing conditions and ∼132 kDa in its native form as analyzed by SDS-PAGE and gel filtration chromatography, respectively. N-terminal amino acid sequencing showed highly conserved residues in a glutamine-rich motif related to binding substrate. The enzyme exhibited optimal activity towards L-Leu at pH 8.5, and like other reported SV-LAAOs, it is stable until 55 °C. Kinetic studies showed that the cations Ca2+, Mg2+ and Mn2+ did not alter Bpic-LAAO activity; however, Zn2+ is an inhibitor. Some reagents such as ß-mercaptoethanol, glutathione and iodoacetate had inhibitory effect on Bpic-LAAO activity, but PMSF, EDTA and glutamic acid did not affect its activity. Regarding the biological activities of Bpic-LAAO, this enzyme induced edema in mice (MED = 7.8 µg), and inhibited human platelet aggregation induced by ADP in a dose-dependent manner and showed antibacterial activity on Gram (+) and Gram (-) bacteria. Bpic-LAAO cDNA of 1494 bp codified a mature protein with 487 amino acid residues comprising a signal peptide of 11 amino acids. Finally, the phylogenetic tree obtained with other sequences of LAAOs, evidenced its similarity to other homologous enzymes, showing two well-established monophyletic groups in Viperidae and Elapidae families. Bpic-LAAO is evolutively close related to LAAOs from B. jararacussu, B. moojeni and B. atrox, and together with the LAAO from B. pauloensis, form a well-defined cluster of the Bothrops genus.

Caracterización de la enzima similar a trombina del veneno de Bothrops pictus "jergón de costa" / Characterization of thrombin like enzyme from Bothrops pictus venom

Realizar una caracterización bioquímica y molecular del principio coagulante del veneno de Bothrops pictus. Materiales y métodos. Se realizó la amplificación del gen a partir de cDNA, se analizó la homología de la secuencia nucleotídica y de la proteína deducida. Se procedió a purificar la enzima para los análisis de secuenciación directa N terminal de los primeros 20 aminoácidos y los ensayos de coagulación sobre plasma humano y fibrinógeno humano, por otro lado, se evaluó el patrón de corte del fibrinógeno por medio de PAGE SDS y la actividad defibrinogenante en roedores albinos (18-22 g). Se determinó el contenido de carbohidratos asociados, el efecto de inhibidores clásicos de proteasas y el efecto de iones bajo la forma de cloruros. Resultados. La enzima mostró homología en la estructura primaria con otras TLEs reportadas para la familia Viperidae, la dosis coagulante mínima (DCM) sobre plasma y fibrinógeno humano fue de 18 y 6 ug respectivamente y su potencia coagulante fue de 131,1 NHI unidades de trombina. La enzima se mostró estable a condiciones fisiológicas y prescinde de iones para su actividad. Los carbohidratos asociados detectados fueron hexosas (25,76%), hexosaminas (13,1%) y ácido siálico (0,76%). Los agentes fluoruro de fenil metil sulfonil floruro (PMSF) ditiotreitol (DTT) fueron los principales inhibidores de la actividad enzimática en tanto que la heparina no tuvo efecto inhibidor. Conclusiones. El principio coagulante del veneno de Bothrops pictus es una enzima similar a trombina...

To perform a biochemical and molecular characterization of the coagulant principle from Bothrops pictus venom. Materials and methods. We amplified the genetic sequence of this enzyme from cDNA and analyzed the homology of its nucleotide sequence and its deduced protein. This enzyme was also purified for N-terminal sequencing of first 20 amino acids and for coagulation assays using human plasma and human fibrinogen. Furthermore, cleavage pattern on fibrinogen was evaluated using SDS-PAGE and defibrinogenant activity on white mice (18-22 g). Finally, associated carbohydrate content, effect of protease inhibitors and chloride ions on its enzymatic activity were analyzed. Results. The Thrombin-like Enzyme from Bothrops pictus showed homology at primary level of structure with other previously reported TLEs from Viperidae family. Minimum Coagulant Dosis (MCD) on plasma and human fibrinogen were 18 and 6 ug, respectively, and its coagulant potency was 131.1 NHI Thrombin units. This TLE was stable under physiological conditions and chloride ions are not necessary for its activity. Detected associated carbohydrates were hexoses (25.76%), hexosamines (13.12%) and sialic acid (0.76%). Phenyl methyl sulphonyl fluoride (PMSF) and dithiothreitol (DTT) were the main inhibitors of its enzymatic activity, but heparin had no inhibitor effect. Conclusions. The coagulant principle of Bothrops pictus venom is a Thrombin-like enzyme...

[Characterization of thrombin like enzyme FROM Bothrops pictus venom]. / Caracterización de la enzima similar a trombina del veneno de Bothrops pictus "jergón de costa.

OBJECTIVES: To perform a biochemical and molecular characterization of the coagulant principle from Bothrops pictus venom. MATERIALS AND METHODS: We amplified the genetic sequence of this enzyme from cDNA and analyzed the homology of its nucleotide sequence and its deduced protein. This enzyme was also purified for N-terminal sequencing of first 20 amino acids and for coagulation assays using human plasma and human fibrinogen. Furthermore, cleavage pattern on fibrinogen was evaluated using SDS-PAGE and defibrinogenant activity on white mice (18-22 g). Finally, associated carbohydrate content, effect of protease inhibitors and chloride ions on its enzymatic activity were analyzed. RESULTS: The Thrombin-like Enzyme from Bothrops pictus showed homology at primary level of structure with other previously reported TLEs from Viperidae family. Minimum Coagulant Dosis (MCD) on plasma and human fibrinogen were 18 and 6 µg, respectively, and its coagulant potency was 131.1 NHI Thrombin units. This TLE was stable under physiological conditions and chloride ions are not necessary for its activity. Detected associated carbohydrates were hexoses (25.76%), hexosamines (13.12%) and sialic acid (0.76%). Phenyl methyl sulphonyl fluoride (PMSF) and dithiothreitol (DTT) were the main inhibitors of its enzymatic activity, but heparin had no inhibitor effect. CONCLUSIONS: The coagulant principle of Bothrops pictus venom is a Thrombin-like enzyme.

Coagulant thrombin-like enzyme (barnettobin) from Bothrops barnetti venom: molecular sequence analysis of its cDNA and biochemical properties.

The thrombin-like enzyme from Bothrops barnetti named barnettobin was purified. We report some biochemical features of barnettobin including the complete amino acid sequence that was deduced from the cDNA. Snake venom serine proteases affect several steps of human hemostasis ranging from the blood coagulation cascade to platelet function. Barnettobin is a monomeric glycoprotein of 52 kDa as shown by reducing SDS-PAGE, and contains approx. 52% carbohydrate by mass which could be removed by N-glycosidase. The complete amino acid sequence was deduced from the cDNA sequence. Its sequence contains a single chain of 233 amino acid including three N-glycosylation sites. The sequence exhibits significant homology with those of mammalian serine proteases e.g. thrombin and with homologous TLEs. Its specific coagulant activity was 251.7 NIH thrombin units/mg, releasing fibrinopeptide A from human fibrinogen and showed defibrinogenating effect in mouse. Both coagulant and amidolytic activities were inhibited by PMSF. N-deglycosylation impaired its temperature and pH stability. Its cDNA sequence with 750 bp encodes a protein of 233 residues. Indications that carbohydrate moieties may play a role in the interaction with substrates are presented. Barnettobin is a new defibrinogenating agent which may provide an opportunity for the development of new types of anti-thrombotic drugs.